Rabies is a disease that can occur in all warm-blooded species and is caused by rabies virus. Infection with rabies virus followed by the outbreak of the clinical features in nearly all instances results in death of the infected species. In Europe, the USA and Canada wild life rabies still exists and is an important factor in the cause of most human rabies cases that occur. On the other hand, urban rabies constitutes the major cause of human rabies in developing countries.
Rabies virus is a non-segmented negative-stranded RNA virus of the Rhabdoviridae family. Rabies virus virions are composed of two major structural components: a nucleocapsid or ribonucleoprotein (RNP), and an envelope in the form of a bilayer membrane surrounding the RNP core. The infectious component of all Rhabdoviruses is the RNP core which consists of the RNA genome encapsidated by the nucleocapsid (N) protein in combination with two minor proteins, i.e. RNA-dependent RNA-polymerase (L) and phosphoprotein (P). The membrane surrounding the RNP core consists of two proteins: a trans-membrane glycoprotein (G) and a matrix (M) protein located at the inner site of the membrane.
The G protein, also referred to as spike protein, is responsible for cell attachment and membrane fusion in rabies virus and additionally is the main target for the host immune system. The amino acid region at position 330 to 340 (referred to as antigenic site III) of the G protein has been identified to be responsible for the virulence of the virus, in particular the Arg residue at position 333. All rabies virus strains have this virulence determining antigenic site III in common.
RABORAL V-RG® was developed as an alternative rabies vaccine by Merial, Ltd. As an alternative rabies vaccine that proved to have the unique and novel attribute of being effective by the oral route (reviewed by Mackowiak et al., Adv Vet Med. 1999; 41:571-83). The vaccine comprises a modified live vaccinia virus containing the rabies surface glycoprotein gene inserted in its genome. The first experimental use of the recombinant vaccine in wildlife was initiated in Europe. The vaccine was contained within a plastic sachet surrounded by an edible bait and deployed into areas known to contain rabies-infected red fox populations. These campaigns resulted in a dramatic reduction in rabies cases in red foxes and the use of RABORAL V-RG® was considered a success. RABORAL V-RG® was also found to be effective in causing a reduction in rabies in raccoons, coyotes and red foxes (reviewed by Mackowiak et al., Adv Vet Med. 1999; 41:571-83), as well as skunks and mongooses (US2005/0282210A1, Maki J L et al, Merial Limited; Grosenbaugh D A, Maki J L et al. J Wildlife Dis 2007; 43(1):124-8).
Despite the success of oral vaccination of wildlife, Rabies continues to pose a significant health threat, killing over 55,000 people annually and necessitating prophylactic treatment of over 15 million people post-exposure each year (WHO: Rabies vaccines). One of the goals of the USDA-APHIS Wildlife Services National Rabies Management Program (WS NRMP) has been to control and eventually eliminate terrestrial rabies in the United States. In contrast to some other parts of the world, the primary reservoir for the virus in the United States is wildlife. In an attempt to achieve rabies eradication, oral rabies vaccines are distributed by hand and aircraft in most states east of the Appalachian Mountains as well as portions of Ohio, Arizona, and Texas. It has been estimated that a vaccination rate of 70% is considered sufficient to break disease transmission cycles (Hethcote, H W et al. 1978). Presently, it is estimated that Oral Rabies Vaccination (ORV) programs in the U.S. successfully vaccinate only 30% of raccoons (Slate, D. et al. 2009), while vaccination rates have been sufficiently high to eradicate gray fox and canine rabies in foxes and coyotes, respectively in Texas (Fearneyhough, M G et al., 1998; Sidwa, T J et al., 2005).
The ORV program led by WS NRMP currently uses the vaccine, RABORAL V-RG® (Merial Ltd., Athens, Ga., described above) in its vaccination campaigns. Currently, RABORAL V-RG® is delivered in a liquid form that is contained within a flavor-coated, plastic sachet. Under optimal conditions, when the sachet is pierced by an animal bite the vaccine is released into the buccal cavity and coats the mucosal lining of the mouth. There the recombinant virus expressing the rabies glycoprotein attaches and enters host cells, triggering an immune response. Previous studies have shown that an approximate dose of 107.7 TCID50/1.5 mL of RABORAL V-RG® is necessary to protect a majority of raccoons from challenge with a wild-type rabies strain (Grosenbaugh, D A et al. 2007). However, because the vaccine is delivered in a liquid form, it is often spilled or rejected by the animal, there are concerns related to the amount of RABORAL V-RG® an animal actually ingests (Grosenbaugh, D A et al., 2007; Jojola, S M et al. 2007).
There are at least two potential mechanisms to increase the effectiveness of orally administered RABORAL V-RG®: increase the viscosity of the vaccine and/or include an adjuvant. Adjuvants are compounds that, when combined with a vaccine antigen, increase the immune response to the vaccine antigen as compared to the response induced by the vaccine antigen alone. Among strategies that promote antigen immunogenicity are those that render vaccine antigens particulate, those that polymerize or emulsify vaccine antigens, methods of encapsulating vaccine antigens, ways of increasing host innate cytokine responses, and methods that target vaccine antigens to antigen presenting cells (Nossal, 1999, In: Fundamental Immunology. Paul (Ed.), Lippincott-Raven Publishers, Philadelphia, Pa.; Vogel and Powell, 1995, In: Vaccine Design. The Subunit and Adjuvant Approach. Powell and Newman (Eds.), Plenum Press, NY, N.Y. p. 141). Because of the essential role adjuvants play in improving the immunogenicity of vaccine antigens, the use of adjuvants in the formulation of vaccines has been virtually ubiquitous (Nossal, 1999, supra; Vogel and Powell, 1995, supra; see also PCT publication WO 97/18837, the teachings of which are incorporated herein by reference).
Conventional adjuvants, well-known in the art, are diverse in nature. They may, for example, consist of water-insoluble inorganic salts, liposomes, micelles or emulsions, i.e. Freund's adjuvant. Other adjuvants may be found in Vogel and Powell, 1995, mentioned supra. Although there is no single mechanism of adjuvant action, an essential characteristic is their ability to significantly increase the immune response to a vaccine antigen as compared to the response induced by the vaccine antigen alone (Nossal, 1999, supra; Vogel and Powell, 1995, supra). In this regard, some adjuvants are more effective at augmenting humoral immune responses; other adjuvants are more effective at increasing cell-mediated immune responses (Vogel and Powell, 1995, supra); and yet another group of adjuvants increase both humoral and cell-mediated immune responses against vaccine antigens (Vogel and Powell, 1995, supra). In sum, adjuvants generally appear to exert their effects in at least one of five ways: 1) facilitate antigen uptake, transport and presentation in the lymph nodes, 2) prolong antigen presentation, 3) signal pathogen-recognition receptors (PRRs) expressed on innate immune cells, 4) cause damage or stress to cells, which signals an immune response, and 5) induce a preferential Th1 or Th2 response (Schijns V E et al. 2007).
Some adjuvants have demonstrated particular adjuvanting utility, in part, by promoting improved absorption through mucosal linings. Some examples include MPL, LTK63, toxins, PLG microparticles and several others (Vajdy, M. Immunology and Cell Biology (2004) 82, 617-627), but many of these are not practical to employ as part of a wildlife vaccination strategy due to the high expense and/or poor stability in the field. One reasonably affordable mucosal adjuvant, chitosan (Van der Lubben et al. 2001; Patel et al. 2005; Majithiya et al. 2008; U.S. Pat. No. 5,980,912), has demonstrated some efficacy when used in certain human vaccines, but the polymer has not previously been successfully combined with veterinary biologics. Persons skilled in the vaccine development arts understand that effective adjuvant/antigen/host combinations are unpredictable and require significant experimentation to derive. See for example Edelman, “An Update on Vaccine Adjuvants in Clinical Trial,” Aids Research and Human Retroviruses 8(8):1409-1411 (1992); McElrath, “Selection of potent immunological adjuvants for vaccine construction,” seminars in Cancer Biology 6:375-385 (1995); Aucouturier et al., “Adjuvants designed for veterinary and human vaccines,” Vaccine 19:2666-2672 (2001); East et al., “Adjuvants for New Veterinary Vaccines,” Chapter 1 in Progress in Vaccinology, vol. 4 Veterinary Vaccines, Springer Verlag, NY 1993, pp 1-28; Altman et al., “Immunomodifiers in Vaccines,” Advances In Veterinary Science and Comparative Medicine 33:301-343 (1989); and Willson et al., “Tissue Reaction and Immunity in Swine Immunized with Actinobacillus pleuropneumoniae Vaccines,” Can J Vet Res 59:299-305 (1995).
In view of the current suboptimal vaccination of wildlife against pathogens such as rabies, there is real and long-felt need for an adjuvanted vaccine bait that can be widely distributed at reduced cost and increased efficacy. An ideal adjuvanted bait should 1) be safe, inexpensive and easy to formulate, 2) reduce the amount of antigen dose required to achieve effective protection, 3) be relatively more viscous, to reduce loss/spillage, and 4) be stable in the environment long enough for the animals to become effectively vaccinated.